BACKGROUND

Since the introduction of solid phase peptide synthesis (SPPS) by Merrifield in 1963 (3), the technique has been used in the production of many peptides in small and large quantities. The first stage of the technique consists of peptide chain assembly with protected amino acid derivatives on a polymeric support. The second stage of the technique is the cleavage of the peptide from the resin support with the concurrent cleavage of all side chain protecting groups to give the crude free peptide.

Sometimes, strategy is chosen that cleaves side chain protecting groups prior to or after the removal of the peptide from the solid support. The third stage of the process is purification of the crude cleaved product. In Merrifield's first report, he employed a beaded form (200 to 400 mesh) of a nitrated chloromethylated divinylbenzene-styrene co-polymer as a solid phase support and N-carbobenzyloxy (Z) groups for alpha-amino acid protection. Z groups were removed with 10 % hydrogen bromide in acetic acid, and the peptide was cleaved from the support by saponification.

In subsequent reports by Merrifield and others following his approach,2-5 divinylbenzene-styrene co-polymers were used that were chemically stable to chain assembly but labile to anhydrous mineral acid cleavage conditions. He employed for amino protection the N-t-butyloxycarbonyl (Boc) group and benzyl ester or ether protection for aspartic and glutamic acids or for serine and threonine. This approach is commonly termed Boc / benzyl chemistry as opposed to the Sheppard approach. Sheppard's modifications employed 9-fluorenylmethyloxycarbonyl (FMOC) amino acid derivatives and peptide to support linkages cleavable with relatively mild acid reagents (trifluoroacetic acid) for peptide cleavage. The cleavage method that has predominated Boc / benzyl chemistry since introduction by Sakakibara in 1965 (6) employs anhydrous liquid hydrogen fluoride (HF) as a cleavage reagent. The HF cleavage method is difficult for the large scale manufacture of peptides because of the hazardous nature of HF.

The need to develop a process for the manufacture thymosin a1 as a therapeutic agent prompted a review of literature for a cleavage strategy for SPPS technology. This review revealed a recent report by H. Yajima and co-workers (5) on the use of hard acid cleavage reagents. This report discussed the use of trifluoromethanesulfonic acid (TFMSA), its trimethylsilyl ester (TMSOTF) and bromotrimethylsilane (TMSBr) with thioanisole as a cation acceptor in trifluoroacetic acid (TFA) solution.

After a careful review of this report, we decided to investigate the work with TMSBr because it appeared that this reagent was well suited for the thymosin alpha 1 peptide resin and of these three reagents TMSBr was reported to give the least rearrangement product of aspartic acid to the isomeric beta-derivative. This is an important consideration for thymosin alpha1 synthesis since it contains three aspartic acid residues as well as a carboxyl terminal asparagine residue. We have extended this cleavage procedure to PAM resins and shown the usefulness of the cheaper trimethyl silyl chloride.

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